CZ2000444A3 - Process for preparing insulin C-peptide - Google Patents

Abstract

A method of producing insulin C-peptide which comprises expression a multimeric polypeptide comprising multiple copies of a Cpeptide insulin, in host cells, and cleavage of this of the expressed polypeptide in which it is released insulin C-peptide copy. Nucleic Acid Molecules expression vectors and host cells for use in such and a multimeric insulin C-peptide polypeptide expressed and cleaved by this procedure.

Description

Process for the production of insulin C-peptide

Technical field

The present invention relates to the production of insulin C-peptide from recombinant DNA molecules comprising multimeric copies of a gene sequence encoding said insulin C-peptide.

BACKGROUND OF THE INVENTION

Insulin is a protein hormone that is involved in the regulation of blood sugar. Insulin is produced in the liver in the form of a proinsulin precursor, which consists of chains of insulin B and A linked together via a C-peptide (hereinafter referred to as the pro-insulin C-peptide referred to as "insulin C-peptide"). chain B and A. Several recent studies have shown that the C-peptide is clinically relevant (Johansson et al., Diabetologia (1992) 35, 121-128 and J.Cin.Endocrinol. Metab. (1993) 77, 976-981). In patients suffering from type I diabetes lacking endogenous C-peptide, administration of this peptide improves renal function, stimulates muscles and glucose utilization, and improves hematoretinal barrier function (Johansson et al., 1992 and 1993 supra).

Although not yet widely recognized, medical circles are increasingly aware of the therapeutic utility of insulin C-peptide. Therefore, there is a need for an easy, economical and efficient synthesis of the insulin C-peptide. While methods of chemical peptide synthesis, for example by sequential addition of amino acids on a solid support, are now well developed, these methods are, despite automation, time-consuming and, more importantly, expensive; a certain limitation also applies to the maximum length of the maximum length. A peptide that can be synthesized economically and reliably. The expression of recombinant DNA appears to be an alternative method of producing peptides, but this method also has disadvantages, e.g. in terms of yield.

The current procedure for the production of insulin C-peptide is based on the processing of proinsulin, a precursor of insulin and C-peptide, usually using trypsin and carboxypeptidase B (Nilsson et al. (1996), J. Biotechnol. 48, 241-250; Jonasson et al. , (1996) Eur J Biochem 236, 656-661). Proinsulin was expressed in the form of a fusion protein allowing a high level of expression in E. coli, wherein the fusion protein was constructed in such a way that the proteolytic processing of proinsulin into insulin and C-peptide simultaneously cleaved the partner to which proinsulin was fused. Proinsulin was prepared as a fusion protein with a ZZ segment derived from staphylococcal protein A that binds IgG (immunoglobulin G) (Nilsson et al., (1987), Prot. Eng. 1, 107-113). This tag was chosen for fusion for reasons of stability to proteolysis, IgG binding ability, high attainable expression levels, and solubilizing properties. The chosen production strategy allowed the use of the affinity tag for efficient purification after solubilization of the inclusion bodies and subsequent renaturation, without the need for further separate operations to cleave and remove the affinity tag; this was cleaved simultaneously with the digestion of proinsulin by the trypsin / carboxypeptidase B pair to form insulin and C-peptide. However, the production of small peptides through the expression of large fusion proteins generally provides rather low yields since the resulting product represents only a small portion of the expressed gene product.

Shen describes in Why. Nati. Acad. Sci. USA, 81, 4627-4631 (1984), a method for preparing human proinsulin by expressing a fused or unfused gene product comprising a gene product containing fusion or non-fusion gene products. _And multiple tandem-linked copies of the proinsulin polypeptide domain. ≪ tb > ______________________________________ < tb > ______________________________________ < tb > From this gene product, the individual proinsulin units are obtained by cyanogen bromide cleavage, but the next step is to generate insulin by cleavage of the proinsulin units by trypsin and caboxypeptidase.

Thus, there remains a need for a recombinant expression method that would increase the yield of insulin C-peptide as an unfused product. The present invention responds to this need.

The invention seeks to improve current methods for recombinant expression of peptides and is essentially based on the idea of increasing the amount of target peptide expressed (in this case, the C-peptide of insulin) by expressing it as part of a single gene product, a multimer (i.e., a multimeric polypeptide). containing multiple copies of the target peptide (insulin C-peptide), and subsequently such a multimeric gene product (i.e., a multimeric polypeptide) would be cleaved to release the target peptide as separate monomeric units.

SUMMARY OF THE INVENTION

Thus, in one aspect, the invention provides a method of producing an insulin Cpeptide comprising expressing a multimeric polypeptide comprising multiple copies of said insulin C-peptide in a host cell and cleaving the expressed polypeptide to release individual copies of the insulin C-peptide (i.e., release of C-peptide monomers) insulin from a multimer).

A multimeric polypeptide (a gene product) is encoded by a genetic construct (in other words, a nucleic acid molecule) that contains multiple copies of a nucleotide sequence that encodes a C-

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insulin peptide. Multiple copies, or tandem repeats, are linked in a recombinant molecule in such a way that they are transcribed and translated together in a single multimeric gene product (ie, a multimeric polypeptide), ie, in a "read format"; for example, multiple nucleotide sequences are linked in a single reading frame within the recombinant molecule. In essence, it is preferred that the genetic construct comprises a concatemer of a nucleotide sequence encoding an insulin C-peptide. Preferably, the genetic construct comprises tandem copies of the coding nucleotide sequences. The recombinant molecule so constructed is then introduced into a host cell and expressed in a standard manner. The expressed gene product (polypeptide) may then be isolated and cleaved to release the C-peptide insulin monomers.

Thus, in another aspect, the invention provides a method of producing C15 insulin peptides, comprising culturing a host cell comprising a nucleate acid molecule comprising multiple copies of a nucleotide sequence encoding said insulin C-peptide, wherein the culturing is carried out under conditions where multimeric is expressed from said nucleate molecule. polypeptide, and cleaving said expressed polypeptide to release individual copies of said insulin C-peptide.

The term "multiple" or "multimeric" as used herein refers to two or more copies of the C-peptide of insulin or nuclic acid encoding it, preferably 2 to 50, 2 to 30 or 2 to 20, more preferably 2 to 15 , or 2 to 10. Other exemplary ranges also include 3 to 20, 3 to 15, or 3 to 10.

The construct may advantageously contain 3 or more copies, eg 3 to 7, or 5 to 7 copies of the nucleotide sequence encoding the C-peptide. ···· insulin. A range of 7 copies or more, for example 7 to 30, 7 to 20 or 7 to 15 may also be useful.

The term "insulin C-peptide" as used herein includes all forms of insulin C-peptide, including natural or synthetic peptides. Such insulin C-peptides may be human peptides or may come from other animal species and genera, preferably mammals. Thus, variants and modifications of the native insulin C-peptide are included as long as they retain the activity of the insulin C-peptide. The insulin C-peptides can be expressed in natural form, i. as various allelic variants, as naturally occurring in different species or due to geographical variations, etc., or as functionally equivalent variants or derivatives thereof, which may differ in amino acid sequence, for example by truncation (e.g. at the N- or C-terminus or at both ends) or other amino acid deletions, insertions, or substitutions. Modifications of protein or peptide sequences while maintaining their useful activities are known in the art; standard techniques known in the art and described in the literature are used for this purpose, e.g., random or site-directed mutagenesis, nucleic acid cleavage and ligation, etc. Functionally equivalent variants or derivatives of natural insulin C-peptide sequences can thus be readily prepared by techniques well known in the art. in the art, and include peptide sequences having functional, e.g., biological activity, of the natural C-peptide insulin. Such insulin C-peptide activities include, for example, stimulation of Na ⁺ K ⁺ ATPase, which may be the basis of various therapeutic activities attributed to C-peptide, eg in the treatment of diabetes or in the treatment or prevention of complications accompanying diabetes such as diabetic neuropathy, nephropathy and retinopathy . Fragments of natural or synthetic insulin peptides may also have desirable functional properties of the peptides from which they are derived and are therefore also included. Particular mention may be made of the insulin C-peptide fragments described by Wahren et al. In WO98 / 13384. All such analogs, variants, derivatives or fragments of the insulin C-peptide are included within the scope of the invention and are included within the term "insulin C-peptide".

Preferably, natural human C-peptide insulin can be used, and this is shown in Figs. 2C (SEQ ID NO: 1).

In another preferred embodiment of the method of the invention, the gene construct further comprises a sequence coding for a partner with which the product is fused, for example a partner (affinity tag) capable of binding to carriers used in processing the gene expression product.

The term "fusion partner" refers to any protein or peptide or derivative or fragment thereof that is translated in sequence with the insulin peptide and whose properties can be utilized in the further processing of the expressed fusion product.

The interaction of the partner to which the C-peptide of insulin is fused with the carrier can be based on affinity, chelating peptides, hydrophobic or electrostatic interactions, or any other mechanism known in the art. The partner to which the insulin C-peptide is fused is preferably one of a pair of affinity-binding molecules or ligands, e.g., a protein, polypeptide or peptide, capable of selectively or specifically binding or reacting with the ligand. Suitable fusion partner molecules include, for example, streptococcal protein G and staphylococcal protein A and derivatives thereof, β-galactosidase, glutathione-S-transferase and avidin or streptavidin, or a fragment or derivative of any of said proteins having a strong affinity for immunoglobulin G, respectively. substrate analogs or antibodies, respectively biotin. Such interactions can be utilized in the purification of fusion protein products from the complex mixture. The ZZ protein A fragment (see Nilsson et al., Supra) is an example of a proteinaceous protein. • a fragment that could be used. Histidine peptides can be used in the fusion as they bind to metal ions, such as Zn ²⁺ , Cu ²⁺ or Ni ^2+, and elution is achieved by lowering the pH or by EDTA (Ljungquist et al. (1989) Eur. J. Biochem. 186, 563-569). A particularly preferred polypeptide partner for fusion is the 25 kDa serumalbumin (BB) binding region derived from streptococcal protein G (SpG) (Nygren et al. (1988) J. Mol. Recogn. 1, 69-74) or other SpG-derived albumin-binding labels (Download and Nygren (1997) Path. Bio. 45, 66-76). Oberg et al., Describes the pTrp BB expression vector (SEQ ID NO: 14) suitable for inserting gene fragments to express the desired product in fusion with BB (Proceedings of the 6 ^th European Congress on Biotechnology, 1994, 179-182). These polypeptides have a strong affinity for albumin, and therefore, purification of the expressed fusion protein can be based on affinity chromatography, eg using an albumin-bearing column. The albumin is preferably immobilized on a solid support.

The cleavage step, i.e. cleavage of monomeric insulin C-peptides from the multimeric polypeptide, i.e. from the expressed gene product and optionally from the partner to which the product is fused, if present, can be accomplished by any convenient means. Enzymes are preferred for this purpose. The primary gene expression product, i.e., the multimeric polypeptide or fusion product or fusion protein, which contains multiple copies (monomers) of the insulin C-peptide fused to the partner is preferably cleaved in one step by one or more proteolytic enzymes to produce unfused single copies of C- of an insulin peptide. A particularly preferred way to obtain the desired cleavage products is by co-treatment with trypsin and carboxypeptidase B (e.g., bovine, porcine, or other sources). Trypsin cleaves proteins behind each arginine residue (C-terminal) and carboxypeptidase B removes the C-terminal arginine present on

any tryptic peptide resulting from trypsin digestion. The conditions for achieving proteolytic cleavage are well known in the art, as are a variety of other suitable proteolytic enzymes such as subtilisin (including mutants thereof), enterokinase, factor Xa, thrombin, IgA protease, protease 3C, and inteins. For example, it has been found that 60 min incubation of the expressed gene product with proteolytic enzymes (e.g. trypsin and carboxypeptidase B) is sufficient to fully process the expressed protein. Preferably, an incubation period of 5 minutes may be sufficient to adequately process the fusion protein such that no fusion or multimeric protein is detectable by conventional SDS-PAGE. In an alternative method, the starting gene expression product is cleaved with chemical reagents such as CNBr, hydroxylamine or formic acid.

Depending on the exact nature of the nucleic acid molecule and insulin C-peptide used (genetic construct), cleavage sites, eg for proteolysis, may be naturally present or may be introduced by artificially manipulating the genetic construct using known techniques, eg site-directed mutagenesis. ligation of nucleotide sequences encoding the appropriate cleavage site, etc.

The multimeric expressed polypeptide may preferably comprise a linker, i. a linking amino acid residue or linking peptide comprising or providing a cleavage site. Preferably, the cleavage site comprises a cleavable motif that is recognized and cleaved by a proteolytic enzyme. Linker regions may be included between the individual "monomer" peptides in the multimer product and / or optionally also between the fusion partner, if present, and the monomer peptide. Preferably, each peptide monomer may be tandem aligned with a linker region. Preferably, the C-peptide insulin monomers in the multimer are preferably flanked by suitable

linker sequences providing for cleavage and release of the insulin C-peptide without any linker region residues. The linker region may contain 1 to 15, e.g. 1 to 12 or 1 to 10 amino acid residues, but the length is not critical and may be selected at will. Linker regions of 1 to 8, e.g., 1, 5 and 7 residues may be preferred. The individual linker regions in a given construct may be the same or different, although they are usually the same in practical terms. Thus, for example, linkers beginning or ending with arginine residues may be used to cleave the combined action of trypsin and carboxypeptidase B.

Alternatively, the linker comprises the amino acid lysine, either alone or as part of a longer sequence, and can also be cleaved by the trypsin / carboxypeptidase B combination.

Such linkers for inclusion among the insulin C-peptide monomers may preferably begin and end with a cleavage site, e.g., an arginine residue at both the N- and C-termini, to allow release of the insulin C-peptide monomer without additional amino acids. When linking a linker between a fusion partner and / or the C-peptide multimer end of an insulin insulin, a single cleavage site (eg, Arg) may be present at the appropriate end of the linker (or analogously at the appropriate cleavage site). ).

Exemplary typical linker regions include -RTASQAR- (SEQ ID NO: 2) for inclusion among C-peptide monomers, -ASQAR- (SEQ ID NO: 3) between the C-peptide and fusion partner multimer, and -RTASQAVD (SEQ ID NO). : 4) to the end of the multimeter.

As mentioned above, standard procedures well known in the art can be used to introduce linker sequences.

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Another aspect of the invention is a nucleic acid molecule comprising multiple copies of a nucleotide sequence encoding an insulin C-peptide, wherein the nucleic acid molecule encodes a multimeric polypeptide capable of cleaving into individual copies of said insulin C-peptide.

In another aspect, this aspect of the invention can be perceived to provide a nucleic acid molecule that comprises a concatemer of a nucleotide sequence that encodes an insulin C-peptide.

Various aspects of the invention set forth above (and below) include embodiments in which the multimeric polypeptide (gene product) does not contain insulin A and insulin B peptides, or wherein the nucleic acid molecule does not encode insulin A and insulin B peptides. the number of copies of the insulin C-peptide in the multimeric polypeptide, or encoded by the nucleic acid molecule, is equal to two, the multimeric polypeptide does not include, or the nucleic acid molecule does not encode the insulin A and B peptides.

In a particularly preferred embodiment of the invention, the nucleic acid molecule will additionally comprise a nucleotide sequence that encodes a fusion partner that aids in further processing the encoded multimeric polypeptide, for example, that is useful in purifying the expressed protein product. The gene encoding the fusion partner will be in the correct position and orientation to be translated together with multiple copies of the insulin C-peptide to form a single fused peptide initially. Suitable proteins for fusion are discussed above.

Preferably, the nucleic acid molecule will also comprise one or more nucleotide sequences that encode linker regions that contain cleavage sites as discussed above.

Thus, as examples of nucleic acid molecules of the invention, those that encode the polypeptide of (I) may be mentioned.

H ₂ N - A - (CX) n - COOH (I) where

C is an insulin C-peptide;

A is a bond, or F, wherein F is a fusion partner, or - (F-X) -;

X is a linker region comprising at least one cleavage site, wherein each X is the same or different; and n is an integer from 2 to 50.

This aspect of the invention encompasses an embodiment wherein formula (I) includes the condition that for n = 2 said polypeptide (I) does not contain an insulin A and B chain.

The insulin C-peptides (group C), fusion partners (group F) and linker regions (group X) may be as defined above. Similarly, n can be as defined above in relation to the terms "multiple" and "multimeric".

Preferably, the nucleic acid molecule or genetic construct useful in the use of the invention will contain suitable regulatory sequences that will regulate expression in the host cell. Such expression control sequences include, for example, transcriptional regulatory elements (eg, promoter-operator regions, ribosome binding sites, termination signals, enhancer sequences, etc.), and translational regulatory elements (eg, initiation and stop codons) linked in the corresponding reading frame. to coding sequences.

Any suitable host cells, including prokaryotic and eukaryotic cells, are useful and are selected according to the chosen expression system, eg, bacterial, yeast, insect (eg baculovirus-based) or mammalian. Many different expression systems are known and described in the art. For example, E. coli may be used as host cells for peptide production, in which case the regulatory sequences may comprise, for example, the E. coli trp promoter. Other suitable hosts include Gram-negative bacteria other than E. coli, Gram-positive bacteria, yeast, insect, plant or animal cells, eg, genetically manipulated cell lines.

Expression vectors comprising the nucleic acid molecules described above represent another aspect of the invention.

Any preferred vector can be used to achieve expression by the method of the invention, many of which are known in the art and described in the literature. Suitable vectors thus include plasmids, cosmids or vectors derived from viruses. However, these vectors which are introduced into host cells for expression are preferably plasmid, phage or viral vectors. The vectors may contain suitable regulatory sequences linked in a corresponding reading frame to the nucleic acid molecules of the invention. Other genetic elements, eg, replicons or sequences facilitating or facilitating transfer of the vector to the host cell, stabilizing functions, eg, helping to maintain the vector in the host cell, cloning sites, restriction endonuclease cleavage sites, or marker coding sequences may also be included using techniques well known in the art. . The vectors may persist in the host cell as separate entities or, as in the case of plasmid insertion vectors or other insertion vectors, may be incorporated into the chromosome of the host cell. Random non-specific integration into the host chromosome is contemplated, but specific homologous integration is more preferred. Techniques suitable for this purpose are known in the art (see, eg, Pozzi et al. (1992) J. Res. Microbiol. 143, 449-457 and (1996) Gene 169, 85-90). Integration is "homologous" because the plasmid insertion vector contains a segment of chromosomal DNA of the host cell.

Exemplary typical plasmids suitable for expression of the genetic constructs or nucleic acid molecules of the invention include pTrpBB (Öberg et al., Supra) or derivatives thereof. Such plasmids may optionally be modified, if desired, to remove fusion partner coding sequences. Any high copy number vector containing a Trp-promoter or the like can be used.

A variety of techniques well known in the art may be used to introduce such vectors for expression into prokaryotic or eukaryotic cells, e.g., bacterial transformation, transfection, electroporation techniques. Transformed or transfected eukaryotic or prokaryotic host cells, i.e., host cells comprising a nucleic acid molecule of the invention and as defined above, represent another aspect of the invention.

As described in more detail in the Examples, expression vectors, particularly plasmids carrying the nucleic acid molecules of the invention, are advantageous in that they exhibit genetic stability in their hosts; as assessed by restriction mapping, no genetic instability was detected in plasmids prepared from cell cultures grown to high densities.

Another aspect of the invention provides a method of producing a nucleic acid molecule that encodes a multimeric polypeptide comprising multiple copies of an insulin C-peptide, wherein the expressed multimeric polypeptide is capable of producing single copies of the insulin C-peptide after cleavage, said method comprising forming a nucleic acid molecule comprising multiple copies of the nucleotide sequence encoding the insulin C-peptide, joined in the corresponding reading frame.

Numerous techniques are known in the art for generating multimeric copies of a gene or gene fragment that can be used in the methods of the invention. For example, synthetic DNA fragments can be polymerized in a head-to-tail fashion, using single-stranded non-palindromic overhangs designed for this purpose. The polymerized DNA fragments can then be directly ligated with the corresponding overhangs resulting from restriction enzyme digestion (Ljungquist et al. (1989) Eur. J. Biochem. 186, 563-569). Other methods for achieving gene mutation are based on the use of restriction enzymes of the ILS type, such as Bsp M1 (Stáhal et al. (1990) Gene 89, 187-193) or Bsm I (Haydn and Mandecki (1988) DNA 7, 571-). 577). Alternative strategies utilize gene fragment polymerization and ligation of adapter molecules containing restriction sites that allow further subcloning (Aslund et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1399-1403 and Irving et al. (1988) in Technological Advances in Vaccine Development, AR Liss Inc., New York, pp. 97-105). Methods using polymerase chain reaction (PCR) for de novo gene synthesis that would be useful for preparing multimeric gene fragments are also known (Majumder (1992) Gene 110, 89-94 and Nguyen et al. (1994) in Advances in Biomagnetic Separation) , Eaton Publishing Co., Natick, pp. 73-78).

In a preferred embodiment of the method of the invention, the purified gene fragments (i.e. nucleotide sequences encoding the insulin C-peptide) are allowed to be polymerized head-to-tail (multimerized) via the proposed non-palindromic methods.

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The protruding ends are then ligated into a plasmid opened by a restriction enzyme, preferably the enzyme Sfi I.

In a particularly preferred embodiment, a plasmid comprising a nucleotide sequence (e.g., a gene fragment) encoding the insulin C-peptide is digested with a restriction enzyme and said sequence or gene fragment is removed and ligated back into the digested plasmid after multimerization of the sequence or gene fragment. Transformant colonies can advantageously be analyzed by a PCR technique (Stahi et al. (1993) Biotechniques 14, 424-434), in which a segment encoding one or more copies of the insulin C-peptide is also amplified. The size of the fragments amplified in the PCR reaction can be compared by agarose gel electrophoresis. In another preferred embodiment, gene fragments encoding the desired number of concatemerized insulin C-peptides, eg, three or seven, are isolated and ligated into another plasmid that has been digested with the same restriction enzyme used to cleave the insulin C-peptide fragment. This plasmid, which will be used to transform the host cells, most preferably comprises a suitable promoter and sequences encoding a suitable partner for fusion with the insulin C-peptide.

Other aspects of the invention include the products of the above methods, namely the insulin C-peptide multimer and the individual C-peptides released by cleavage from the multimer.

In particular, this aspect of the invention provides a multimeric polypeptide comprising multiple copies of an insulin C-peptide that is cleavable to form individual copies of said insulin C-peptide. Optionally, the multimeric polypeptide may additionally comprise a fusion partner and / or linker regions comprising a cleavage site flanking each said C-peptide monomer.

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Also provided is a method of producing a multimeric polypeptide comprising multiple copies of an insulin C-peptide that is cleavable into discrete copies of said insulin C-peptide, the method comprising culturing a host cell comprising a nucleic acid molecule encoding said multimeric polypeptide under conditions such as the multimeric polypeptide expressed, and recovering the expressed multimeric polypeptide.

Host cells can be cultured by techniques known in the art, eg, batch or continuous.

The multimeric gene product or polypeptide can be obtained from a host cell culture by standard techniques well known in the art, eg, by standard cell lysis and protein purification techniques. As mentioned above, if a fusion partner is included in the multimeric polypeptide, easy purification can be achieved based on the affinity binding of that partner.

Numerous techniques are known in the art for isolating proteins or polypeptides from cells or culture media, both natural and recombinantly expressed, and any of them can be used. Cell lysis to release intracellular proteins / polypeptides can be accomplished by any of a variety of methods known in the art and described in the literature and, if necessary, additional purification steps can be used, again based on techniques known in the art, depending on whether batch or continuous culture methods were used.

In the case of the multimeric C-peptide insulin polypeptides of the invention, methods of lysing cells and recovering polypeptides by heat treatment have proved to be particularly effective, for example, the method described in WO90 / 00200 and modifications thereof. Such methods include heating the culture medium with the host cells to, for example, a temperature of 50-100 ° C for a period of time, generally not exceeding 1 hour, with the addition of: The expressed polypeptide is released into the medium, preferably in substantially pure form. This is believed to be due to the selective release of the expressed polypeptide. It has been observed that this method works well in the case of soluble, thermally stable, polypeptide products, whether recombinant or not (thus, the method may have general applicability), but has proven to be particularly useful for multimeric C-peptide insulin polypeptides according to of the invention where surprisingly high product yields in high purity can be obtained. For example, such heat treatment can be accomplished by heating to 80100 ° C, e.g. 85-99 ° C or 90-95 ° C, for 5-20 minutes, e.g. 8-10 minutes, and then cooling, e.g., to a temperature of 0-4 ° C or cooling on ice.

After isolation of the multimeric polypeptide, it can be cleaved to release the individual monomers of the insulin C-peptide. Thus, another aspect of the invention provides a method of producing an insulin C-peptide, the method comprising cleaving a multimeric polypeptide as defined above to release individual copies of said insulin Cpeptide.

After cleavage of the multimeric polypeptide to release the individual C-peptide monomers as discussed above, these can also be further purified, eg, to homogeneity (eg, by SDS-PAGE) using well known standard purification techniques, such as ultrafiltration, gel filtration, clarification, reverse phase chromatography, etc.

Another aspect of the invention is the therapeutic use of the cleaved peptide products obtained by the methods described above. Cleaved C-peptide insulin can be used in the treatment of type 1 diabetes and / or complications associated with diabetes. Accordingly, within the scope of the invention

Also contemplated is a method of treating type 1 diabetes or its complications, which comprises administering the C-peptide of insulin, prepared by any of the methods described above.

The invention will now be described in more detail by way of non-limiting Examples and with reference to the following figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 schematically shows the construction of recombinant plasmids of the invention, including multimerization of a gene fragment encoding a C-peptide.

Figure 2A and B schematically show two gene products, BB-C3 (A) and BB-C7 (B). The linker regions that surround the C-peptide are indicated by a single letter code. Arginine residues (printed in bold) flank each C-peptide.

Figure 2C shows the amino acid sequence of the C-peptide in a single letter code (SEQ ID NO; 1).

Figure 3 is a copy of a photograph of SDS-PAGE (10-15%) gel under reducing conditions and shows the BB-C3 fusion protein (lane 1) and the BB-C7 fusion protein (lane 2) after affinity purification on HSASepharose. Molecular weight standards, 94, 67, 43, 30, 20 and 14 kDa, are in lane M.

Figure 4A - is a copy of a photograph of SDS-PAGE (10-15%) gel under reducing conditions and shows BB-C3 after incubation with trypsin and carboxypeptidase Β. Lane 1 contains uncleaved fusion proteins, Lane 2 contains protein digests after 5 minutes of trypsin and carboxypeptidase působení. In lane M, the molecular weight standards are 94, 67, 43, 30, 20 and 14 kDa.

• - • - - - - - - - - - - - - - -

Figure 4Β - is the same as Figure 4A except that it relates to the BB-C7 fusion product.

Figure 5 shows the analytical separation of equimolar cleavage products of BB-C7 (top) and BB-C3 (middle) with trypsin and carboxypeptidase B in reverse phase chromatography (RPC). Insulin C-peptide (Sigma) was used as control (below).

Figure 6 shows superimposed gel filtration chromatograms (Superdex Peptide, Pharmacia Biotech, Uppsala, Sweden) of BB-C7 fusion product digested with trypsin (weight ratio 5000: 1) and carboxypeptidase B (weight ratio 2000: 1) for the indicated time.

Figure 7 - shows analysis of C-peptide of insulin derived from BB-C7 (A) compared to Eli Lilly (B) or Sigma (C) insulin C-peptide standards in reverse phase chromatography.

Figure 8 shows the amino acid sequence (in single letter code) of a peptide product containing a BB fusion partner, and seven copies of the insulin C-peptide (SEQ ID NO: 5).

Figure 9 shows SDS-PAGE (10-15%) analysis of synthesized BB-C1 (lane 1), BB-C3 (lane 2) and BB-C7 (lane 3) fusion proteins after affinity purification on HSA-Sepharose. The sizes of the molecular weight standards are indicated in kDa.

Figure 10 shows RPC (reverse phase chromatography) analysis of products of equimolar cleavage of BB-C1 and BB-C3 and BB-C7 respectively with trypsin + carboxypeptidase B. A commercially available C-peptide standard (see below) was used as a control (see below). Sigma).

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Figure 11 shows electrophoretic analysis (in agarose gel, 1%) of Kpn1-PstI grafts of pTrpBB-C7 plasmid preparations obtained from E. coli cultured with 0 (lane), 7 (lane 2), 27 (lane 3) or 31 hours (lane) 4). In lane 5, the Kpn1-Pst1 digest of the original pTrpBB-C7 plasmid used to transform E. coli cells is shown, and in lane 6, pTrpBB-C7 is uncleaved after 31 hours of culture. In lanes M (size standards), lambda phage DNA is digested with PstI. The arrow indicates the position of the C7 fragment.

Figure 12 shows SDS-PAGE analysis (under reducing conditions) of BB-C7 culture samples. Lane 1: 2μ (culture medium. Lane 2: 0.5 μΙ of sonicated culture. Lane 3: 0.5 μΙ medium after heat treatment of the culture. The arrow indicates the position of BB-C7 fusion protein. In lane M the molecular weight standards are 94, 67 , 43, 30, 20 and 14 kDa.

DETAILED DESCRIPTION OF THE INVENTION

Example 1 - Construction of recombinant DNA

Four synthetic Jope 10 oligonucleotides (5'-CGGCCTCCCA GGCCCGCGAA GCTGAGGACC TGCAAGTTGG TCAGGTTGAA

CTGGGCGGTG GCCCGGGTGC AGGC-3 ') (SEQ ID NO. 6), Jope 11 (5'-TCTTTGCAGC CGCTGGCTTT AGAAGGTTCT CTTCAGCGTA CGGCCTCCCA

CTGGACGTTC AACCAGTCCA ACTTGACCCG CCACCGGG-5 ') (SEQ

ID NO: 8) and Jope 13 (3'-CCCACGTCCG AGAAACGTCG)

GCGACCGAAA TCTTCCAAGA GAAGTCGCAT GCCGGAGGGT CCGGCAGCTG ATTG-5 ') (SEQ ID NO: 9) were phosphorylated and Jope 10: Jope 12 and Jope 11: Jope 13 pairs were hybridized by incubation. * «Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί 70 ·· ·· at 70 ° C for 10 min followed by cooling to room temperature. The two linkers thus formed were mixed and ligated into plasmid pUC18 (Yanish-Perron et al., 1985, Gene 33, 103-106) opened by restriction enzyme digestion Κρη I -Pst I (Fig. 1) and dcm 'cells were transformed with the ligation mixture. strains of Escherichia coli GM31 (Marinus, 1973, Mol. Gen. Genet. 127, 47-55). A transformant (pUC-C1) containing the correct nucleotide sequence in the cloned gene fragment encoding the C-peptide of insulin was identified by a solid-phase DNA sequencing technique using PCR (Hultman et al. Io (1989) Nucl. Acids Res. 17, 4937-4946) . Plasmid DNA was prepared from pUC-C1 and after digestion with Sfi I, both the cleaved fragment encoding the insulin C-peptide and the vector portion were purified using the Mermaid (glass-milk) kit (BIO 101 Inc, CA, USA). ) and GeneClean kits (BIO 101 Inc, CA, USA).

The purified insulin C-peptide gene fragments were polymerized head-to-tail by designing non-palindromic overlapping ends and then ligated back into the purified Sfi I open vector. E. coli RRIAM15 cells (Ruther (1982) Nucl. Acids Res. 10, 5765-5772) were transformed with the ligation mixture and the resulting transformants were analyzed using an adapted PCR technique (Stáhal et al. (1993) supra). Briefly, individual colonies were picked into PCR vials containing 50 μΙ PCR reaction mixtures (20 mM TAPS, pH 9.3 at 20 ° C,

2 mM MgCl ₂ , 50 mM KCl, 0.1% Tween-20, 0.2 mM dNTP, after 6 pmoles of both primers (RIT27: 5'-GCTTCCGGCTCGTATGTGTG-3 '(SEQ ID NO: 10) and RIT28: 5' -AAAGGGGGATGTGCTGCAAG GCG-3 '(SEQ ID NO: 11) and 1.0 Taq polymerase unit). Both PCR primers RIT27 and RIT28 have hybridization sites in pUC18 and flank the site into which fragments of insulin C-peptide are introduced. PCR amplified fragments from clones containing different numbers of oligonucleotides introduced were compared to each other (as opposed to pUC18) using agarose gel electrophoresis to identify transformants carrying one to seven inserts. The resulting plasmids were designated pUC-C1, pUC-C2 etc.

Gene fragments containing the required number of inserts were excised from the plasmid preparations using the enzymes Κρη I - Pst I. Fragments encoding one, three, or seven concatomerized insulin C-peptides were isolated and ligated into similarly cleaved pTrpBBT1T2 and the resulting plasmids were designated pTrpBB-C1 and pTrpBB-C3 and pTrpBB-C7, respectively. Plasmid pTrpBBT1T2 was constructed from the plasmid pTrpBB (Öberg et al. (1994) in Proc. 6 ^th Eur. Congress).

Biotechnol., Elsevier Science B.V., pp. 179-182) by introducing a transcription terminator sequence derived from plasmid pKK223-3 (Pharmacia Biotech, Uppsala, Sweden). The transcription terminator sequence was obtained from pKK223-3 using a standard PCR amplification protocol (Hultman et al. (1989) supra} and HEAN-19 oligonucleotides,

5'-CCCCCTGCAGCTCGAGCGCCTTTA ACCTGTTTTGGCGGATG-3 '(SEQ ID NO: 12) and HEAN-20, 5'-CCCCAAGCTTAGAGTTTGTAG AAACGC-3' (SEQ ID NO: 13).

The restriction sites introduced by the PCR reaction were digested with Pst I and Hind III 25 and the fragment inserted into pTrpBB digested with the same enzymes.

The resulting expression vector encodes an affinity "holder" consisting of an eight amino acid leader sequence derived from the trp operon and a BB-binding serumalbumin (25 kDa) region (Nygren et al. (1988) supra) derived

The transcription is regulated by the E. coli trp promoter. The plasmid further carries a kanamycin resistance gene.

Example 2 - Protein Expression and Purification

E. coli cells carrying pTrpBB-C3 and pTrpBB-C7 and thus encoding BB-C3 or BB-C7 fusion proteins were grown overnight at 37 ° C on a shaker in flasks containing 10 ml Tryptic Soy Broth (Difco, USA) ( 30 g / l) with the addition of yeast extract (Difco) (5 g / l) and kanamycin monosuifate (50 mg / ml). The overnight cultures were diluted 10-fold and 100 ml of this diluted culture was grown in shake flasks in the same medium at 37 ° C. Gene expression was induced at mid-log phase (A ₆ oo = 1) by addition of β-indole acrylic acid at a concentration of 25 mg / l. Cells were harvested 20 hours after induction by centrifugation for 10 min at about 6000 g. The cells were resuspended in 1/20 culture volume in TST (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 0.05% Tween 20, 1 mM). EDTA), lysed by sonication and centrifuged at about 40,000 g. Samples for sonication were prepared by centrifuging the culture and resuspending the cells in 30 ml cold TST buffer. Samples made

2o were held on ice during 2-minute sonication pulses, sonication was performed on a Vibra Cell (500W) from Sonics and Materials Inc. (Danbury, Connecticut, USA) using a 13 mm standard conical extension, 70% duty cycle (20 kHz), and power set to 6.5. Supernatants containing soluble proteins were filtered (0.45 μιη) and diluted to 100 ml with TST buffer. Soluble fusion proteins were isolated by affinity chromatography on HSA-Sepharose (human serum albumin) (Nygren et al. (1988) supra) as described by Stáhal et al. (1989) J. Immunol. Meth. 124, 43-52. In eluted • 4

4 ·· ·

The protein fraction was determined by absorbance at 280 nm and the relevant fractions were lyophilized.

Figure 3 shows affinity purified BB-C3 and BB-C7 proteins, respectively, after one purification step on HSA-Sepharose. Both fusion proteins were dominated by full-length products whose electrophoretic mobility corresponded to their molecular weights, 39.1 and 54.2 kDa, respectively. Expression levels in shake flask cultures were almost identical for both fusion proteins: 130 mg / L for BB-C3 and 120 mg / L for BB-C7.

Example 3 - Proteolytic cleavage of fusion proteins

Trypsin, which cleaves behind the basic amino acid residues (i.e., C-terminally), has been used for a long time to cleave fusion proteins. Despite the expected low specificity, trypsin has been shown to be useful for the specific cleavage of fusion proteins by leaving basic residues within the folded protein domains without cleavage (Wang et al. (1989) J. Biol. Chem. 264, 21116-21121). Trypsin has the added advantage of being cheap and readily available. Here, trypsin was used in combination with carboxypeptidase B to process fused BB-C3 resp. BB-C7 to obtain a native human C-peptide insulin. Thus, trypsin will cleave the fusion proteins C-terminally after each arginine residue, and carboxypeptidase B will then remove the C-terminal arginine present on each insulin C-peptide monomer after trypsin digestion.

To analyze the cleavage efficiency, both fusion proteins, BB-C3 and BB-C7, were incubated with trypsin and carboxypeptidase B for two hours.

and then analyzed by SDS-PAGE. It was found that both fusion proteins were processed rapidly and no fusion protein was seen in SDS-PAGE after 5 minutes of cleavage (Fig. 4A and B).

In addition to this analysis, the relative yields of insulin C-peptide monomer after cleavage of the BB-C3 fusion proteins, respectively, were compared. BB-C7. The mixtures after treatment with trypsin and carboxypeptidase B on equimolar amounts of BB-C3, respectively. BB-C7 were analyzed by reverse phase HPLC (250 mm, Kromasil C8 column, 4.6 mm ID, 7 µιτι particle size, Hewlett Packard 1090). A 10-40% gradient of acetonitrile in 0.1% trifluoroacetic acid over 30 min at 40 ° C was used to elute. As shown in FIG. 5, the cleavage of the BB-C7 fusion protein resulted in a significantly higher ratio of the C-peptide insulin product (elution time ca. 25.4 min) to the other cleavage products (derived from the BB portion) compared to the cleavage of the BB-C3 fusion protein. Integration of peak areas corresponding to the insulin C-peptide (C7: C3) gave a peak area ratio of 2.43, a value close to the theoretical value of 2.33.

However, this does not imply any information about the completeness of fusion protein processing. To determine when the treatment with trypsin and carboxypeptidase B reaches completeness, the time dependence of the enzymatic cleavage of the BB-C7 fusion protein was performed. The lyophilized BB-C7 fusion protein was dissolved to a final protein concentration of 1 mg / ml in 100 mM phosphate buffer pH 7.5 containing 0.1% (vol / vol) Tween 20. Trypsin (T-2395, Sigma, St. Louis) , MO, USA) and carboxypeptidase B (Boehringer Mannheim) were added to the resulting trypsin / fusion protein weight ratio of 1/5000 and carboxypeptidase B / fusion protein 1/2000. Mon 15, 30, 60 and 120 · «· · * * * *

26- ’:

minutes and the digestion was stopped by lowering the pH to 3 by addition of acetic acid. Acetonitrile was added to a 20% (vol / vol) concentration to stabilize the cleavage products.

The digestion material was chromatographed by gel filtration (Superdex Peptide Column, SMART ™ system, Pharmacia Biotech, Uppsala, Sweden) and the chromatograms were superimposed (Fig. 6). The analysis revealed that BB-C7 was completely finished after 60 minutes under these conditions, as no further C-peptide insulin was obtained by prolonging the incubation period. These results also suggest that quantitative yields of insulin C-peptide could be obtained from fusion proteins containing multimeric forms of insulin C-peptide.

Example 4 - Characterization of the obtained insulin C-peptide:

Reverse phase chromatography (RPC) and mass spectroscopy

Two different assays were performed to confirm that the peptide obtained indeed corresponds to the native human C-peptide insulin. In the first, reverse phase chromatography (RPC) was used to compare the RPC-purified insulin C-peptide obtained by cleavage of BB-C7 with the insulin C-peptide standards, using the C-peptide obtained from Eli Lilly (CA, USA) as standards. ) and a commercially available insulin peptide, fragment 3-33 (Sigma, USA). C-Peptide Preparations 0 · 7 · 7 · 7 · 7 · 7 · 7 Insulin was analyzed by RPC on a Sephasil C8 5qm SC2, 1/10 column using the SMART ™ system (Pharmacia Biotech, Uppsala, Sweden). A 26-36% gradient of acetonitrile in 0.1% (vol / vol) trifluoroacetic acid over 20 min at 25 ° C was used to elute. The flow rate was 100 μΙ / min and the absorbance was measured at 214 nm. The analysis showed that all three preparations were almost identical, having the same retention times and the same low impurity content (Fig. 7). In a second analysis, the insulin C-peptide obtained from BB-C7 was subjected to mass spectroscopy (Table 1) on a JEOL SX102 mass spectrometer (JEOL, Japan), equipped with an electrospray. Good agreement in weights (Table 1), together with the observed similarity to insulin C-peptide standards in comparative RPC analysis, suggests that native human C-peptide insulin was prepared by recombinant procedure.

Table 1. Molecular weights of insulin C-peptide (Da)

Calculated 3020.3

Experimental 3019.7 ± 1.8

Example 5 - Characterization of the obtained insulin C-peptide monomer: Radioimmunoassay (RIA)

2o The insulin C-peptide monomer obtained by cleavage of the BB-C7 fusion protein was analyzed using a commercially available radioimmunoassay kit, developed to monitor human insulin C-peptide levels, eg, in blood and urine (Euro-Diagnostica, Malmo, Sweden; Cat. MD 315). For comparison, insulin preparation C-peptide (Eli-Lilly Co., Indianapolis, Ind., USA), r 28, whose biological activity has been previously demonstrated (Johansson et al. (1992) Diabetologia 35, 1151-1158) was also analyzed. Samples for analysis were prepared by weighing both preparations of C-peptide and then diluting to a final concentration of 3.31 and 3.31, respectively. 3.30 nmoles / L in solution

0.05 M Na-phosphate buffer, pH 7.4, 5% human serum albumin (HSA) a

0.02% Thimerosal. Briefly, the kit comprises rabbit anti-human C-peptide antiserum, ¹²⁵ L-labeled human C-insulin insulin as tracer, as a secondary goat anti-rabbit anti-Ig antibody in PEG environment, human C-peptide insulin standards and control w samples for construction of calibration. curves to quantify the C-peptide of insulin in the test samples. The results of the analysis of the two samples are summarized in Table 2 below. The results show that in RIA these two preparations are identically recognized and quantified.

Table 2. Comparative RIA analysis of insulin C-peptide with demonstrated biological activity and insulin C-peptide obtained by cleavage of recombinant BB-C7 fusion protein.

sample expected concentration determined concentration (nM) (nM)

Insulin C-peptide (cleavage 3.31 2.34 (71%) BB-C7 fusion protein)

C-peptide of insulin (from Eli-Lilly)

3.30

2.41 (73%) ··· * - - 29 · · · · · · · · · · · · · · · · · · ·

Example 6 - Expression, purification and proteolytic cleavage of BB-C1, BB-C3 and BB-C7 fusion proteins

This Example provides additional comparative results to the experiments in Examples 2 and 3 for the BB-C1 fusion protein.

E. coli cells harboring plasmids pTrp BB-C1 or pTrp BB-C3 or pTrp BB-C7 (see Example 1) were cultured, and fusion proteins expressed, isolated, purified and analyzed as described in Example 2.

Analysis of E. coli transformed pTrp BB-C1 or pTrp BB-C3 or pTrp BB-C7 showed that the encoded fusion proteins BB-C1, BB-C3 and BB-C7 accumulated intracellularly as soluble gene products (data not shown). After cell disruption, the fusion proteins were effectively purified by HSA-affinity chromatography. Giant. 9 shows affinity purified fusion proteins BB-C1, BB-C3 and BB-C7 after one purification step on HSA-Sepharose. All three fusion proteins were dominated by full-length products whose electrophoretic mobility was consistent with their molecular weights: 31.5,

39.1 and 54.2 kDa. Expression levels were reproducible and similar for all three different fusion proteins, ranging from 40-60 mg / L.

To verify the efficacy of the proteolytic processing of these three affinity purified fusion proteins, BB-C1, BB-C3, and BB-C7 were incubated with trypsin and carboxypeptidase B for various times and then subjected to SDS-PAGE analysis. All three fusion proteins were processed rapidly, and after 5 minutes of proteolytic treatment, no full-length fusion protein was detected in SDS-PAGE (data not shown).

The proteolytic cleavage efficiency was further analyzed as described in Example 3 and it was found that BB-C7 was cleaved to completion after 60 minutes.

To more adequately compare the relative yields of C-peptide monomers after cleavage of the BB-C1, BB-C3, and BB-C7 fusion proteins, reverse phase HPLC analysis was performed (as described in the Example).

3). The digested mixtures were analyzed after 120 min of trypsin + carboxypeptidase B for approximately equimolar amounts of BB-C1, BB-C3 or BB-C7 (A220 monitored). The results (Fig. 10) showed a significantly higher ratio of C-peptide to other cleavage products in the case of BB-C7 and BB-C3 fusion proteins, as compared to cleavage of the BB-C1 fusion protein. Approximately equimolar amounts from each fusion protein were loaded onto the RPC column, as seen from the same peak heights corresponding to the tryptic cleavage of the BB portion of the fusion proteins seen in all three chromatograms (Fig. 10). Integration of peak areas corresponding to Cpeptide (940, 2324 and 5647 xsx 10 ^-3 absorbance units for BB-C1, BB-C3 and BB-C7, respectively) yielded a ratio of 2.5 for C3: C1 and 6.0 for C7: C1, which are close to the theoretical values of 3, resp. 7.

The results further show a better yield of insulin C-peptide monomers from the insulin C-peptide multimers (C3, C7) compared to the monomeric fusion protein (C1).

• 4 • 4 «4 4 · • 4 4 4 · 4 4 • · 4 4 · 4 • · 4 * 4 4 4 4 * · _Λ . · 4 * • · · ♦ ·· ^ β ί«. · 4 4

Example 7 - Monitoring of genetic stability of plasmid pTrpBB-C7 encoding BB-C7 fusion protein

This Example describes how the genetic stability of plasmid pTrpBB-C7 encoding the BB-C7 fusion protein was determined. E. coli cells carrying plasmid pTrpBB-C7 were grown for various lengths of time and samples were taken after 0, 7, 27 and 31 hours of culture. 31 hours would correspond to the culture time in the production of BB-C7 by large-scale fermentation. Plasmids were obtained from the samples according to standard procedures (Sambrook et al., A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, New York) and cleaved with the restriction enzymes ηρη I and Pst I the fragment encoding the C7 concatemer (see Fig. 1). As a control, the original plasmid pTrpBB-C7, used to transform E. coli cells, was also subjected to Κρη I and Pst I cleavage. 11, the digested fragment has the same size for all samples, demonstrating that the plasmid pTrpBB-C7 remains genetically stable even over longer cultures.

Example 8 - Thermal treatment results in selective release of BBC7 into the culture medium

The example describes how the release of the BB-C7 fusion protein into the culture medium can be achieved by heat treatment and thereby significantly increase the purity of the starting material for further purification of BB-C7. Compared to the most widespread procedure for the isolation of recombinant proteins produced intraceously in E. coli (including four individual operations consisting of centrifugation, resuspension of a cell pellet in a suitable buffer and cell disruption by high pressure homogenization), the process of releasing a gene product under heat treatment has many advantages: (i) the manufacturing process involving the heat treatment has one centrifugation step less, (ii) the stability of the gene product increases as thermal denaturation of the host proteases occurs, (iii) precipitation of the other E. coli proteins results in a significant initial purification of the gene product; iv) nucleic acid release is reduced compared to procedures involving cell disruption. The method could also be suitable for the release of other intracellularly expressed recombinant proteins that remain soluble even at high expression levels and are stable to the thermal action required to release the protein.

E. coli cells carrying plasmid pTrpBB-C7, which encodes BB-C7, were cultured as described in Example 2. Instead of destroying the cells by sonication (Example 2), a thermal treatment can be used to achieve selective and effective release of BB-C7 into the culture medium. . After completion of the culture, the flask and culture was immersed in a boiling water bath for 8-10 minutes. After this time, the culture reached a temperature of approximately 90 ° C. The flask was then placed on ice. FIG. 12 (lane 3) it can be seen that by heat treatment, the BB-C7 fusion protein was released into the culture medium without releasing a substantial amount of host proteins. They are probably completely denatured by this choking. In contrast, sonication (Fig. 12, lane 2) and other mechanical cell disintegration methods result in the release of all host proteins as well as nucleic acids, making the starting material for further purification of the BB-C7 protein very heterogeneous. Normally, very little protein is secreted by E. coli cells into the medium (Fig. 12, lane 1). BB-C7 was heat resistant and could be further purified and further processed to C-peptide monomers as described in Examples 1-3.

4 «

* ♦ ♦ 4 4 4 4 4 4 4 4 4

LIST OF SEQUENCES (1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Creative Peptides Sweden AB (B) STREET: Dahlbergsstigen 6 (C) CITY: Djursholm (E) COUNTRY: Sweden (F) Postcode (ZIP): S-182 64 (i) APPLICANT:

(A) NAME: Stefan Stáhal (B) STREET: c / o Royal Institute of Technology (C) CITY: Stockholm (E) COUNTRY: Sweden (F) Postcode (ZIP): S-100 44 (i) APPLICANT:

(A) NAME: Mathias Uhlen (B) STREET: c / o Royal Institute of Technology (C) CITY: Stockholm (E) COUNTRY: Sweden (F) Postcode (ZIP): S-100 44 (i) APPLICANT:

(A) NAME: Per-Ake Nygren (B) STREET: e / o Royal Institute of Technology (C) CITY: Stockholm (E) COUNTRY: Sweden (F) Postcode (ZIP): S-100 44 (i) APPLICANT:

(A) NAME: Per Jonasson (B) STREET: c / o Royal Institute of Technology (C) CITY: Stockholm (E) COUNTRY: Sweden • a a aa · · · · · · · · · · · ·

(F) ZIP Code (ZIP): S-100 44 (ii) NAME OF THE INVENTION: Recombinant expression of insulin C-peptide (iii) NUMBER OF SEQUENCES: 14 (iv) COMPUTER FORMAT:

(A) MEDIA TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOSZMS-DOS (D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (EPO) (2) SEQ ID NO NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) MOLECULA TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Pro 15 10 15

Gly Ala Gly Ser Leu Gin For Leu Ala Leu Glu Gly Ser Leu Gin 20 25 30 (2) INFORMATION ON SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 Amino Acids (B) TYPE: Amino Acid (A) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULA TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Arg Thr Ala Ser Gin Ala Arg 1 5 (2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) MOLECULA TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Ala Ser Gin Ala Arg 15 (2) INFORMATION ABOUT SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear

- 36 (ii) MOLECULA TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Arg Thr Ala Ser Gin Ala Val Asp 1 5 (2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 521 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) MOLECULA TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Lys Ala Ile Phe Val Leu

Ala Asn Phe As Gin Phe Asn Lys Tyr Gly Val Ser Asp Tyr Lys 20 25 30

Asn Asu Asle Asn Asn Ala Lys Thr Val Glu Gly Val Lys Asp Leu Gin 35 40 45

Ala Gin Val Val Glu Ser Llu Lys Ala Arg Ile Ser Glu Ala Thr 50 55 60

Asp Gly Leu Ser Asp Phe Leu Lys Ser Gin Thr

- 37 Val by you Ser Ile Glu Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu 35 90 95

Leu Asp Lys Asr Gly Val Ser Asp Asr His Lys Asn Leu Ile Asn 100 105 110

Ala Lys Thr Val Glu Gly Val Lys Asp Leu Gin

Ser Ala Lys Lys Ala Ala Arg Ile G Ser Glu Asp Ala Thr Asp Gly Leu Asp Ser Asp 130 135 140

Phe Leu Lys Ser Gin Thr

145 150 155 ISO

Leu Ala Glu Ala Lys Val Leu Ala Gn Asu Arg Glu Leu Asp Lys Tyr Gly

165 170 175

Val Ser Asp Tyr Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu 180 185 190

Gly Val Lys Alu Leu Glu Ile Leu Ala Ala Leu Pro Lys Thr 195 200 205

Asp Thr Tyr Lys Leu 220

Thr Thr Glu

(+420) 225 230 235 240

Ile Leu Glu Asn Ser Ser Ser Val Ala Ser Gin Ala Arg Glu Ala

245 250 255

Glu Asp Glu Gly Glu Glu Glu Glu Gly Gly Gly Gly Pro

260

265

270

- 38 • ·

Gly Ser Leu Gin Glu Ser Leu Gin Arg Thr Ala 275 280 285

Ser Gin Ala Glu Glu Glu Le Gin Glu Gin Glu Glu Leu 290 295 300

Gly Gly Gly Pro Gly Ala Gly Ser Leu Gin Pro Leu Ala Leu Glu Gly

305 310 315 320

Ser Leu Gin Thr Ala Ser Gin Ala Glu Ala Glu Asp Leu Gin

(+420) 325 330 335

Val Gly Gin Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gin 340 345 350

Pro Leu Ala Leu Glu Gly Ser Leu Gin Arg Thr Ala Ser Gin Ala Arg 355 360 365

Glu Ala Glu Asp Glu Val Gly Glu Val Glu Glu Glu Glu Pro 370 375 380

Gly Ala Gly Ser Leu Gin For Leu Glu Ala Gly Ser Leu Gin Arg

385 390 395 400

Thr Ala Gin Glu Gin Glu Gin Asp Glu Gin Val Gly Gin Val

405, 410, 415

Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gin Pro Leu Ala Leu 420 425 430

Glu Gly Ser Leu Gin Arg Thr Ala

Le Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser 450 455 460 · · 45 450 45 45 45 45 45 45 45 450 455 460 · · · · · · · · · · · · · · · · · · · · · ·

Leu Gin For Leu Ala Leu Glu Gly Ser

(+420) 465 470 475 480

Ala Glu Glu Ala Glu Le Gin Val Gly Gin Val Glu

485 490 495

Gly Pro Gly Ala Gly Pro Le Gin Pro Leu Ala Glu Gly Ser Leu 500 505 510

Gin Arg Thr Ala Ser Gin Ala Val Asp 515 520 (2) INFORMATION ON SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 74 base pairs (B) TYPE: nucleic acid (C) STRING TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = “oligonucleotide '(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

CGGCCTCCCA GGCCCGCGAA GCTGAGGACC TGCAAGTTGG TCAGGTTGAA CTGGGCGGTG

GCCCGGGTGC AGGC (2) INFORMATION ON SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 68 bp (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded

-40 (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = “oligonucleotide” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

TCTTTGCAGC CGCTGGCTTT AGAAGGTTCT CTTCAGCGTA CGGCCTCCCA GGCCGTCGAC 60

TAACTGCA 68 (2) INFORMATION ON SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 68 bp (B) TYPE: nucleic acid (C) STRING TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

CATGGCCGGA GGGTCCGGGC GCTTCGACTC CTGGACGTTC AACCAGTCCA ACTTGACCCG SO

CCÁCCGGG ⁶⁸ (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 74 pairs (B) TYPE: nucleic acid (C) STRING TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CCCACGTCCG AGAAACGTCG GCGACCGAAA TCTTCCAAGA GAAGTCGCAT GCCGGAGGGT 60

CCGGCAGCTG ATTG 74 (2) INFORMATION ON SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 bp (B) TYPE: nucleic acid (C) STRING TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

GCTTCCGGCT CGTATGTGTG (2) INFORMATION ON SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 pairs of baffles (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded

- 42 (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

AAAGGGGGAT GTGCTGCAAG GCG (2) INFORMATION ON SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 bp (B) TYPE: nucleic acid (C) STRING TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

CCCCCTGCAG CTCGAGCGCC TTTAACCTGT TTTGGCGGAT G (2) INFORMATION ON SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 pairs of baffles (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear

(ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

CCCCAAGCTT AGAGTTTGTA GAAACGC (2) INFORMATION ON SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4646 base pairs (B) TYPE: nucleic acid (C) STRING TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = “vector” (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

GCGGCCGCTA ATTCATGCTG TGGTGTCATG GTCGGTGATC GCCAGGGTGC CGACGCGCAT 60

CTCGACTGCA CGGTGCACCA ATGCTTCTGG CGTCAGGCAG CCAATCGGAA GCTGTGGTAT 120

GGCTGTGCAG GTCGTAAATC ACTGCATAAT TCGTGTCGCT CAAGGCGCAC TCCCGTTCTG 180

GÁTAATGTTT TTTGCGCCGA CATCATAACG GTTCTGGCAA ATATTCTGAA ATGAGCTGTT 240

GACAATTAAT CATCGAACTA GTGACTAGT ACGCAAGTTC ACGTAAAAAG GGTATCTAGA 300

ATTATGAAAG CAATTTTCGT ACTGAATGCG CAACACGATG AAGCCGTAGA CGCGAATTTC 360

GACCAATTCA ACAAATATGG AGTAAGTGAC TATTACAAGA ATCTAATCAA CAATGCCAAA 420

ACTGTTGAAG GCGTAAAAGA CCTTCAAGCA CAAGTTGTTG AATCAGCGAA GAAAGCGCGT 4 80

ATTTCAGAAG CAACAGATGG CTTATCTGAT TTCTTGAAAT CACAAACACC TGCTGAAGAT 540

ACTGTTAAAT CAATTGAATT AGCTGAAGCT AAAGTCTTAG CTAACAGAGA ACTTGACAAA 600

TATGGAGTAA GTGACTATCA CAAGAACCTA ATCAACAATG CCAAAACTGT TGAAGGTGTA 660

AAAGACCTTC AAGCACAAGT TGTTGAATCA GCGAAGAAAG CGCGTATTTC AGAAGCAACA 720

GATGGCTTAT CTGATTTCTT GAAATCACAA ACACCTGCTG AAGATACTGT TAAATCAATT 780

GAATTAGCTG AAGCTAAAGT CTTAGCTAAC AGAGAACTTG ACAAATATGG AGTAAGTGAC 840

TATTACAAGA ACCTAATCAA CAATGCCAAA ACTGTTGAAG GTGTAAAAGC ACTGATAGAT 900

GAAATTTTAG CTGCATTACC TAAGACTGAC ACTTACAAAT TAATCCTTAA TGGTAAAACA 960

TTGAAAGGC AAACAACTAC TGAAGCTGTT GATGCTGCTA CTGCAAGATC TTTCAATTTC 102 0

CCTATCCTCG AGAATTCGAG CTCGGTACCG GCCTCCCAGG CCCGCGAAGC TGAGGACCTG 10 8 0

CAAGTTGGTC AGGTTGAACT GGGCGGTGGC CCGGGTGCAG GCTCTTTGCA GCCGCTGGCT 114 0

TTAGAAGGTT CTCTTCAGCG TACGGCCTCC CAGGCCCGCG AAGCTGAGGA CCTGCAAGTT 1200

GGTCAGGTTG AACTGGGCGG TGGCCCGGGT GCAGGCTCTT TGCAGCCGCT GGCTTTAGAA 1260

GGTTCTCTTC AGCGTACGGC CTCCCAGGCC CGCGAAGCTG AGGACCTGCA AGTTGGTCAG 13 20

GTTGAACTGG GCGGTGGCCC GGGTGCAGGC TCTTTGCAGC CGCTGGCTTT AGAAGGTTCT 13 8 0

CTTCAGCGTA CGGCCTCCCA GGCCCGCGAA GCTGAGGACC TGCAAGTTGG TCAGGTTGAA 144 0

CTGGGCGGTG GCCCGGGTGC AGGCTCTTTG CAGCCGCTGG CTTTAGAAGG TTCTCTTCAG 1500

CGTACGGCCT CCGAGGCCCG GGAAGCTGAG GAGCTGCAAG TTGGTCAGGT TGAACTGGGC 1560

GGTGGCCCGG GTGCAGGCTC

GCCTCCCAGG CCCGCGAAGC

CCGGGTGCAG GCTCTTTGCA

CAGGCCCGCG AAGCTGAGGA

GCAGGCTCTT TGCAGCCGCT

GTCGACTAAC TGCAGCTCGA

TGATACAGAT TAAATCAGAA

GTAGCGCGGT GGTCCCACCT

ATGGTAGTGT GGGGTCTCCC

AAGGCTCAGT CGAAAGACTG

CTGAGTAGGA CAAATCCGCC

TGGCGGGCAG GACGCCCGCC

ACGGATGGCC TTTTTGCGTT

GCCACGTTGT GTCTCAAAAT

GAACAATAAA ACTGTCTGCT

AACGGGAAAC GTCTTGCTCG

GGTATAAATG GGCTCGCGAT

GGAAGCCCGA TGCGCCAGAG

TTACAGATGA GATGGTCAGA

TTTGCAGCCG

TGAGGACCTG

GCCGCTGGCT

CCTGCAAGTT ggctttagaa

GCGCTTAACT

CGCAGAAGCG

GACCCCATGC

CATGCGAGAG

GGCCTTTCGT

GGGAGCGGAT

ATAAACTGCC

TCTACAAACT

CTCTGATGTT

TACATAAACA

AGGCCGCGAT

AATGTCGGGC

TTGTTTCTGA

CTAAACTGGC

CTGGCTTTAG

CAAGTTGGTC

TTAGAAGGTT

GGTCAGGTTG

GGTTCTCTTC

GTTTTGGCGG

GTCTGATAAA

CGAACTCAGA

TAGGGAACTG

TTTATCTGTT

TTGAACGTT3

AGGCATCAAA

CTAAGCTTTG

ACATTGCACA

GTAATACAAG

TAAATTCCAA

AATCAGGTGC

AACATGGCAA

TGACGGAATT

AAGGTTCTCT

AGGTTGAACT

CTCTTCAGCG

AACTGGGCGG

AGCGTACGGC

ATGAGAGAAG

ACAGAATTTG

AGTGAAACGC

CCAGGCATCA

GTTTGTCGGT

CGAAGCAACG

TTAAGCAGAA

GTGCAGGGGG

AGATAAAAAT

GGGTGTTATG

CATGGATGCT

GACAATCTAT

AGGTAGCGTT

TATGCCTCTT

TCAGCGTACG

GGGCGGTGGC

TACGGCCTCC

TGGCCCGGGT

CTCCCAGGCC

ATTTTCAGCC

CCTGGCGGCA

CGTAGCGCCG

AATAAAACGA

GAACGCTCTC

GCCCGGAGGG

GGCCATCCTG

GGGGGGGAAA

ATATCATCAT

AGCCATATTC

GATTTATATG

CGATTGTATG

GCCAATGATG

CGGACCATCA

1620

1680

1740

1800

1860

1920's

1980

2040

2100

2160

2220

2280

2340

2400

2460

2520

2580

2640

2700

• · * 4 * t X 9 »* · · And t • · • » ♦ 1 · 1 * ······· · • · - 46 »· · · · • · • · · · · · · · B · • · AGCATTTTAT CCGTACTCCT GATGATGCAT GGTTACTCAC CACTGCGATC CCCGGGAAAA 2760 CAGCATTCCA GGTATTAGAA GAATATCCTG ATTCAGGTGA AAATATTGTT GATGCGCTGG 2820 CAGTGTTCCT GCGCCGGTTG CATTCGATTC CTGTTTGTAA TTGTCCTTTT AACAGCGATC 2880 GCGTATTTCG TCTCGCTCAG GCGCAATCAC GAATGAATAA CGGTTTGGTT GATGCGAGTG 2940 ATTTTGATGA CGAGCGTAAT GGCTGGCCTG TTGAACAAGT CTGGAAAGAA ATGCATAAAC 3000 TTTTGCCATT CTCACCGGAT TCAGTCGTCA CTCATGGTGA TTTCTCACTT GATAACCTTA 3060 TTTTTGACGA GGGGAAATTA ATAGGTTGTA TTGATGTTGG ACGAGTCGGA ATCGCAGACC 3120 GATACCAGGA TCTTGCCATC CTATGGAACT GCCTCGGTGA GTTTTCTCCT TCATTACAGA 3180 AACGGCTTTT TCAAAAATAT GGTATTGATA ATCCTGATAT GAATAAATTG CAGTTTCATT 3240 TGATGCTCGA TGAGTTTTTC TAATCAGAAT TGGTTAATTG GTTGTAACAC TGGCAGAGCA 3300 TTACGCTGAC TTGACGGGAC GGCGGCTTTG TTGAATAAAT CGAACTTTTG CTGAGTTGAA 3360 GGATCAGATC ACGCATCTTC CCGACAACGC AGACCGTTCC GTGGCAAAGC AAAAGTTCAA 3420 AATCACCAAC TGGTCCGGAT CCCGGTGCCT CACTGATTAA GCATTGGTAA CTGTCAGACC 3480 AAGTTTACTC ATATATACTT TAGATTGATT TAAAACTTCA ΤΊΤΤΤΑΑΤΤΤ AAAAGGATCT 3540 AGGTGAAGAT CCTTTTTGAT aatctcatga CCAAAATCCC TTAACGTGAG TTTTCGTTCC 3600 ACTGAGCGTC AGACCCCGTA GAAAAGATCA AAGGATCTTC TTGAGATCCT 3660 GCGTAATCTG CTGCTTGCAA ACAAAAAAAC CACCGCTACC AGCGGTGGTT TGTTTGCCGG 3720 ATCAAGAGCT ACCAACTCTT TTTCCGAAGG TAACTGGCTT CAGCAGAGCG CAGATACCAA 3780 ATACTGTCCT 'TCTAGTGTAG CCGTAGTTAG GCCACCACTT 'CAAGAACTCT GTAGCACCGC 3840

- 47 · e e 47

CTACATACCT CGCTCTGCTA ATCCTGTTAC CAGTGGCTGC TGCCAGTGGC GATAAGTCGT 3,900

GTCTTACCGG GTTGGACTCA AGACGATAGT TACCGGATAA GGCGCAGCGG TCGGGCTGAA 3960

CGGGGGGTTC GTGCACACAG CCCAGCTTGG AGCGAACGAC CTACACCGAA CTGAGATACC 4020

TACAGCGTGA GCTATGAGAA AGCGCCACGC TTCCCGAAGG GAGAAAGGCG GACAGGTATC 4080

CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC GCACGAGGGA GCTTCCAGGG GGAAACGCCT 4140

GGTATCTTTA TAGTCCTGTC GGGTTTCGCC ACCTCTGACT TGAGCGTCGA TTTTTGTGAT 4200

GCTCGTCAGG GGGGCGGAGC CTATGGAAAA ACGCCAGCAA CGCGGCCTTT TTACGGTTCC 4260

TGGCCTTTTG CTGGCCTTTT GCTCACATGT TCTTTCCTGC GTTATCCCCT GATTCTGTGG 4320

ATAACCGTAT TACCGCCTTT GAGTGAGCTG ATACCGCTCG CCGCAGCCGA ACGACCGAGC 4380

GCAGCGAGTC AGTGAGCGAG GAAGCGGAAG AGCGCCCAAT ACGCAAACCG CCTCTCCCCG 444 0

CGCGTTGGCC GATTCATTAA TGCAGCTGGC ACGACAGGTT TCCCGACTGG AAAGCGGGCA 4500

GTGAGCGCAA CGCAATTAAT GTGAGTTAGC TCACTCATTA GGCACCCCAG GCTTTACACT 4560

TTATGCTTCC GGCTCGTATG TTGTGTGGAA TTGTGAGCGG ATAACAATTT CACACAGGAA 4620

ACAGCTATGA CCATGATTAC GAATTA 4646

Represented by:

«· ··

- 48 ^. ..... *

P1 / ZOOO-thtyty

Claims (21)

PATENT CLAIMS CLAIMS 1. A method for producing an insulin C-peptide comprising expressing a multimeric polypeptide comprising multiple copies of said insulin C-peptide in a host cell. 5 and cleavage of said expressed polypeptide to release single copies of the insulin C-peptide. 2. A nucleic acid molecule comprising multiple copies of a nucleotide sequence encoding an insulin C-peptide, wherein said nucleic acid also encodes a multimeric polypeptide that can be cleaved to form individual copies of said insulin C-peptide. A method of producing a nucleic acid molecule that encodes a multimeric polypeptide comprising multiple copies of an insulin C-peptide, 15 wherein the expressed multimeric polypeptide can then be cleaved to form single copies of the insulin C-peptide, wherein said method is characterized by comprising forming a nucleic acid molecule comprising multiple copies of the nucleotide sequence encoding the insulin C-peptide that are joined in a corresponding reading frame. 4. A multimeric polypeptide comprising multiple copies of an insulin C-peptide, cleavable to form individual copies of said insulin C-peptide. 5. A method of producing a multimeric polypeptide comprising multiple copies of an insulin C-peptide cleavable to form; said copies of said insulin C-peptide, said method comprising culturing host cells comprising a nuclic acid molecule encoding said multimeric polypeptide, under conditions where the multimeric polypeptide is 5, and isolating the expressed multimeric polypeptide. 6. A method of producing an insulin C-peptide, the method comprising cleaving the multimeric polypeptide of claim 4. 7. The method, nucleic acid molecule or multimeric polypeptide of any one of claims 1 to 6, wherein said multiple copies of an insulin C-peptide or a nucleotide sequence encoding the insulin C-peptide are: 15 tandem arranged. 8. A method, a nucleic acid molecule or a multimeric polypeptide according to any one of claims 1 to 7, wherein said multimeric polypeptide comprises 2 to 30 copies. 20 of said insulin C-peptide. 9. A method, a nucleic acid molecule or a multimeric polypeptide according to any one of claims 1 to 8, wherein said multimeric polypeptide comprises 3 to 7 copies. 25 of said C-peptide insulin. The method, nucleic acid molecule or multimeric polypeptide of any one of claims 1 to 9, wherein the multimeric polypeptide further comprises a partner with which it is fused. 11. The method, nucleic acid molecule or multimeric polypeptide of claim 10, wherein said fusion partner is one of a pair of affinity-binding partners or ligands. The method, nucleic acid molecule or multimeric polypeptide of claim 10 or 11, wherein said fusion partner is a 25 kDa serumalbumin-binding region (BB) derived from streptococcal protein G. 13. The method, nucleic acid molecule or multimeric polypeptide of any one of claims 1 to 12, wherein the insulin C-peptide monomers in said multimeric polypeptide are flanked by linker regions containing cleavage sites. 20 place. 14. The method, nucleic acid molecule or multimeric polypeptide of any one of claims 1 to 13, wherein said cleavage site is cleavable by a proteolytic enzyme. A method, nucleic acid molecule or multimeric polypeptide according to any one of claims 1 to 13, characterized in that: arginine residues for -I i wherein said cleavage site comprises trypsin and carboxypeptidase B cleavage. The method or nucleic acid molecule of any one of claims 1-3 and 7-14, wherein said nucleic acid molecule further comprises one or more regulatory or expression regulatory sequences. An expression vector comprising the nucleic acid molecule of any one of claims 2 and 7-16. The expression vector of claim 17, wherein the vector is a plasmid. 19. An expression vector according to claim 18, characterized in that it is derived from the plasmid pTrpBB (SEQ ID NO: 14) or a derivative thereof. A host cell comprising the nucleic acid molecule of any one of claims 2 and 7-16. An insulin C-peptide produced by the method of claims 1 or 6.